A major task in biochemical and molecular diagnostic laboratories is sample homogenization, cell and tissue lysis and the mixing of reagents. The lysis is used for subsequent isolation of biomolecules in the field of research and development, chemical analysis and diagnostics.
Today, vortexers and bead mills are routinely used to perform these tasks. Such systems agitate the fluids and sample material in a vial in such a way that the vials are accelerated and decelerated in a repetitive pattern in one or more dimensions.
For sample homogenization, beads (glass, ceramic or other materials) are added. Due to the rapid acceleration and deceleration steps, these beads generate a physical impact force on the sample material and ‘grind’ or ‘mill’ it into smallest particles that are then suspended in a surrounding fluid (hence the name ‘bead milling’). On a cellular level, cell walls are cracked under such acceleration, deceleration, impact and shear forces if the bead sizes are selected appropriately. This is for example necessary for DNA tests where access to the DNA embedded in cells is needed. Besides milling with a conventional vortexer, the use of bead mills allows the milling of different materials.
There are also some known chemical agents that improve the cell opening process.
The combination of chemical or enzymatic lysis steps with mechanical milling steps additionally improves the performance of the lysis procedure. The use of chemical or enzymatical steps require certain incubation temperatures, as well as thorough mixing of the reagents during the incubation steps.
Despite the wide usage, the known vortexing and bead milling devices and procedures in many cases have low efficiency and therefore need a substantial amount of processing time.
Additionally, these methods need to be combined with incubation steps at different temperatures to allow efficient lysis of a wide variety of clinical samples. The current methods require a combination of different steps involving the steps of mixing, incubation at different temperatures, ideally interrupted by e.g. bead milling. Each of the steps is performed in different instruments (vortexer, heating block, bead mill . . . ) as a combination of manual steps. That is, these sequential steps require manual work of well trained operators.
In order to avoid the time-consuming operation due to the several instruments needed for a lysis process, efforts have been made to reduce the number of the necessary devices. However, with conventional devices it is not possible to overcome for example problems caused by the high G-forces which occur due to rotation operations (several steps cannot be carried out or cannot be carried out in an appropriate manner during the rotation of the vial, for example appropriate heating and/or determining the temperature of the sample or even temperature regulation of the agitated sample).
With other known methods, the bead milling step is performed at lower temperatures. Several of the bead mills commercially available are equipped with a system which allows the sample to be milled in a refrigerated compartment of the instrument. Other systems allow for submersing the grinding beaker or vial into liquid nitrogen before bead milling, so that the process is carried out at low temperatures. However, such devices do not provide a desired degree of efficiency, especially when treating viscous biological samples.
An analysis of mechanical effects during the bead milling process shows that the efficiency mainly depends on the strength of the impact collisions of the beads with the suspended sample material and cells. Such collision forces can be increased by increasing the density of the beads, the amount of beads and/or increasing the acceleration and deceleration speed. Today's vortexers and bead mills are already at their limits with respect to acceleration and deceleration speeds due to their construction.
A typical known system does not only accelerate and decelerate the sample material and vial but also the much larger masses of the vial holders. Furthermore the movements are most often created by a rotating motor shaft driving an eccentric tappet. This eccentric tappet then drives a plate that is spring suspended for example in the x- and z-axis (that is, right and left movements, up and down movements). The plate is coupled with a vial holder or the vial is pressed on that plate manually. Due to large unbalanced masses, such movements create huge stress on the whole construction, thereby limiting the way of travel (maximum eccentricity/maximum deflection of the vial holder) and the maximum velocities that can be achieved. A typical way of travel is only a few millimeters with a maximum of 3000 to 4000 cycles per minute. To avoid that such systems start moving on the desk surface during operation, they are often equipped with a very heavy additional mass and dampers in the bottomplate.